WO2018183491A1

WO2018183491A1 - Recovery of dried bacteria

Info

Publication number: WO2018183491A1
Application number: PCT/US2018/024836
Authority: WO
Inventors: Adrienne HUNTRESS; William PASUTTI; Ben DOUGHAN; Kimberley CLARKE
Original assignee: Novozymes Bioag A/S
Priority date: 2017-03-30
Filing date: 2018-03-28
Publication date: 2018-10-04

Abstract

Methods are described for recovering certain unculturable bacteria into a culturable form. Bacteria that have been dried, dehydrated, or desiccated, in one example using spray drying, are more efficiently cultivated using media that contains humic acid, salts thereof, analogs thereof, or peat, than when using media that does not contain humic acid or related substances. When humic acid is present in media used in colony-forming-unit (CFU) assays of dried bacteria, higher titers of viable bacteria are obtained than when the same media that does not contain humic acid or related substances is used.

Description

RECOVERY OF DRIED BACTERIA

Background

[0001] Some culturable bacteria can enter nonculturable states but remain viable (Pinto, D. et al., 2015, Thirty Years of Viable but Nonculturable State Research. Critical Reviews in

Microbiology, 41, 61-76). These unculturable states may be caused by various stresses to which the bacteria are exposed, including drying/dehydration/desiccation. Because viable bacteria are historically quantified using colony forming unit (CFU) assays, that require bacteria to grow (i.e., to be culturable), the number of viable bacteria, in populations that contain nonculturable bacteria, are generally underestimated using CFU assays.

Summary

[0002] We have found that non-spore-forming bacteria that are in samples that are dried, dehydrated, or desiccated can be cultivated more efficiently using media that contains humic acid, salts thereof, analogs thereof, or peat, as compared to media that does not contain humic acids or related substances. In some examples, bacteria in spray dried samples form a greater number of colonies in CFU assays when media containing humic acid or related substances is used, as compared to the number of colonies obtained using the same media that does not contain humic acid or related substances. Therefore, for bacteria that have been exposed to drying conditions, viability assays that use bacterial growth as a threshold, provide better estimates of the number of viable cells when humic acid or related substances are included in the culture medium.

Detailed Description

Definitions

[0003] The following includes definitions of selected terms that may be used throughout the disclosure and in the claims. The definitions include various examples and/or forms of components that fall within the scope of a term and that may be used for implementation. The examples are not intended to be limiting. Both singular and plural forms of terms fall within the definitions. [0004] As used herein, "about" means ± 10% with respect to the stated value or parameter.

[0005] As used herein, "agar" means a gelatinous substance, generally derived from seaweed, and used in culture media to provide media that is solid or semisolid in consistency. In some examples, agar concentrations of about 0.5-1.5%) (weight/volume) in media may be used for bacterial culture plates. Herein, agar is considered a type of gelling agent.

[0006] As used herein, "agrochemical" means chemicals used in agriculture like, for example, chemicals used as acaricides, fungicides, gastropodicide, herbicides, insecticides, miticides, and the like.

[0007] As used herein, an "analog" of a first substance (e.g., humic acid) refers to a second substance that is structurally similar to the first substance, but with some differences. An analog may be synthetic.

[0008] As used herein, an "assay" means a test to determine something.

[0009] As used herein, "bacteria" means prokaryotic organisms that have peptidoglycan in their cell walls, and have lipids in their membranes, where the lipids contain fatty acids.

[0010] As used herein, "capable" refers to the ability or capacity to do or achieve a specific thing.

[0011] As used herein, "colony" means a visible cluster of bacteria, generally on the surface of a solid or semisolid medium (e.g., medium containing agar), and probably originating from division of a single cell. A colony formed by bacteria may be called a "bacterial colony" or "colony-forming unit" (CFU).

[0012] As used herein, "contain" means to have or hold. In some examples, when a sample contains or is containing something (e.g., bacteria), the something is within or part of the sample.

[0013] As used herein, "count" when used as a verb, means to tally or total. "Counting" is an act to tally or total. [0014] As used herein, "determine" means to establish or find out. "Determining" is an act to establish or find out. Something that has been established or found out may be said to be "determined."

[0015] As used herein, "dextrose equivalent value" is a measure of the amount of reducing sugars present in a sugar product. The dextrose equivalent value is an indication of the average degree of polymerization for sugars.

[0016] As used herein, "dilution," when used as a noun, refers to a liquid that contains a reduced concentration of a thing as compared to the liquid when undiluted. "Diluting" is an act to create a dilution.

[0017] As used herein, "dissolve," when referring to a solid substance, refers to the solid incorporated into a liquid so as to form a solution.

[0018] As used herein, "dried sample" refers to a sample that has been treated to decrease the moisture content of the sample. When a sample contains bacteria, the bacteria may be referred to as "dried bacteria." Dehydrated or desiccated may be used in place of the word "dried."

[0019] As used herein, "enumerate" means to establish the number of something.

[0020] As used herein, "gelling agent" refers to substances that are added to liquid to cause the liquid to become solid or semisolid in consistency. A variety of these substances exist.

Example gelling agents may include agar, agarose, alginic acid, carrageenan, gelatin, gellan gum, guar gum, xanthan gum, and the like.

[0021] As used herein, "Gram-negative" refers to bacteria that, in a Gram staining reaction, lose the crystal violet stain and take the color of the counterstain.

[0022] As used herein, "humic acid" refers to a principal component of humic substances (fulvic acid and humin are other principal components of humic substances) that is soluble in dilute alkali but which becomes insoluble as the pH becomes acidic. Substances "related to" humic acid may include salts of humic acid, humic acid analogs, synthetic humic acids, and may also include peat. [0023] As used herein, "maltodextrin" refers to a dextrin containing maltose.

[0024] As used herein, "medium," with reference to a culture medium for a bacterium, refers to compositions for supporting growth of bacteria. Example growth medium may include liquid media (e.g., broths) or solid/semisolid media (e.g., agar-containing media).

[0025] As used herein, "moisture content" means the amount of water in a sample. Herein, moisture content is determined on a wet basis (i.e., weight of water in a sample/total weight of sample). For example, a sample with weight 10 grams, 1 gram of which is water, has a moisture content of 0.1 or 10%.

[0026] As used herein, "non-spore forming" refers to bacteria that are not capable of forming spores.

[0027] As used herein, "peat" generally refers to partially decomposed vegetable/plant matter.

[0028] As used herein, "plating" refers to applying a sample, bacteria from a sample, or dilution of the sample or bacteria, to solid or semisolid bacterial culture medium (e.g., agar- containing medium). "Plated" refers to something that has been applied to solid or semisolid bacterial culture medium.

[0029] As used herein, "prepare" means to make something. [0030] As used herein, "prior" means before.

[0031] As used herein, "recover" means to culture a bacterium that is unculturable under another set of circumstances.

[0032] As used herein, "represent" means constitutes or regarded as. [0033] As used herein, "salt" refers to an ionic form of a substance. [0034] As used herein, "sample" refers to a representative part of a whole. [0035] As used herein, "soluble" means able to be dissolved (e.g., in water). "Solubilizing" is an act to dissolve something.

[0036] As used herein, "spray drying" refers to processes that produce dry powders from a liquid or slurry. "Spray dried," when referring to a substance, refers to a substance that has undergone the spray drying process.

[0037] As used herein, "synthetic" refers to something that is synthesized, rather than naturally occurring. A synthetic substance may be an analog.

[0038] As used herein, "unculturable," when referring to a bacterium, means unable to be cultured, using current technologies (i.e., technologies prior to this disclosure; e.g., without humic acid), and generally refers to a certain set of growth conditions (e.g., the medium does not contain humic acid). A bacterium that is considered unculturable may eventually be cultured, for example, when technologies are improved (e.g., with humic acid).

[0039] As used herein, "use" means to employ or put into service. "Using" is an act to employ or put into service. Something that has been employed or put into service may be said to be "used."

[0040] As used herein, "viable" means alive, surviving or living. Dried bacteria

[0041] Samples (generally liquid, but also semi-solid or solid) containing bacteria may be dried, dehydrated or desiccated using a variety of methods. In some examples, a liquid sample may be left open so that moisture from the sample is evaporated into the air. This may be called air drying. In some examples, a gas stream (e.g., air) may apply heat to the sample by convection and moisture/vapor is removed as humidity. In some examples, vacuum drying, where heat is supplied to the sample by conduction, radiation, or microwaves, vapor is produced and carried away by a vacuum system, may be used. In some examples, drum drying, where a surface supplies heat to the sample, vapor is produced and carried away by an aspirator, may be used. Freeze drying or lyophilization may be used in some examples and occurs when the sample is frozen and moisture from the sample is sublimed, generally under a vacuum. In some examples, a dried sample may be produced by draining (e.g., centrifugation to mechanically extract a solvent).

[0042] Spray drying may be used in some examples. Spray drying generally produces small liquid droplets of specific sizes from a liquid or slurry using a spray nozzle or atomizer. After the droplets exit the nozzle or atomizer, they are dried, generally using hot air, to form a powder. Machines known as spray dryers are normally used for this process.

[0043] In some examples, the dry compositions may have a moisture content of less than about 50%, 40%, 30%, 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0044] Drying of samples containing bacteria generally is considered a stress for the bacteria. A variety of stresses may cause bacteria that are culturable to enter an unculturable state.

Stresses like drying, dehydration, and desiccation may cause culturable bacteria to be

unculturable. Likewise, hydration or rehydration of bacteria in a dried, dehydrated, or desiccated sample may be a stress that may cause unculturability.

[0045] For induction of bacteria into an unculturable state using stress, the amount of stress applied to the bacteria may have to be considered. For example, too much stress applied to bacteria (e.g., type of stress, time and/or intensity of the stress) may cause the bacteria to become nonviable and, therefore, not recoverable. Too little stress may fail to place bacteria into an unculturable state at all. There likely is an amount of each different type of stress that places the maximum number of bacteria in a population into an unculturable state. This amount of stress may have to be empirically determined.

[0046] In some examples, viable but unculturable bacteria in a dried sample may become nonviable depending on the time the dried sample is stored (e.g., increasing numbers of viable, unculturable bacteria in the sample may die the longer the bacteria are exposed to the dry state) and/or depending on the conditions (e.g., temperature, relative humidity, and the like) under which the dried sample is maintained.

[0047] The percentage of bacteria within a sample that have entered into an unculturable state may affect the ability of that population to demonstrate recovery (e.g., if fewer bacteria in a population are in an unculturable state, assays that detect recovery of unculturable bacteria to a culturable state, even if robust, may not detect recovery). Hence, even with an assay that can efficiently detect recovery of unculturable bacteria (e.g., humic acid in the medium), there need to be unculturable bacteria in the population in order for the assay to give positive results.

[0048] Many different types of bacteria are known to be capable of entering/exi sting in an unculturable state. Nonlimiting examples of unculturable bacteria that exist in an unculturable state may include γ-proteobacteria, β-proteobacteria, a-proteobacteria, ε-proteobacteria, bacteroidetes, acinobacteria, or firmicutes. In some examples, the bacteria capable of entering/exi sting in an unculturable state include Gram-negative bacteria.

[0049] Nonlimiting examples of unculturable bacteria that exist in an unculturable state may be from the genera Acetobacter, Acinetobacter, Aeromonas, Agrobacterium, Alcaligenes, Arcobacter, Bifidobacterium, Bradyrhizobium, Burkholderia, Campylobacter, Citrobacter, Cytophaga, Enterobacter , Enter ococcus, Erwinia, Escherichia, Francisella, Helicobacter, Klebsiella, Lactobacillus, Legionella, Listeria, Oenococcus, Paracoccus, Pasteurella,

Pseudomonas, Ralstonia, Ramlibacter, Rhizobium, Rhodococcus, Salmonella, Serratia, Shigella, Sinorhizobium, Vibrio, Xanthomonas, and Yersinia.

[0050] Nonlimiting examples of unculturable bacteria that exist in an unculturable state may be Acetobacter aceti, Acinetobacter calcoaceticus, Aeromonas hydrophilia, Aeromonas salmonicida, Agrobacterium tumifaciens, Alcaligenes eutrophus, Arcobacter butzleri,

Bifidobacterium lactis, Bifidobacterium longum, Bifidobacteriumanimalis, Bradyrhizobium japonicum, Bradyrhizobium elkaii, Burkholderia cepacia, Burkholderia pseudomallei,

Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Citrobacter freundii, Cytophaga allerginae, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter agglomerans,

Enterococcus faecalis, Enterococcus hirae, Enterococcus faecium, Erwinia amylovora,

Escherichia coli, Francisella tularensis, Helicobacter pylori, Klebsiella aerogenes, Klebsiella pneumoniae, Klebsiella planticola, Lactobacillus plantarum, Lactobacillus lindneri,

Lactobacillus paracollinoides, Lactobacillus lactus, Legionella pneumophila, Listeria monocyhtogenes, Oenococcus oeni, Paracoccus pantotrophus, Pasteurella piscicida,

Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Ralstonia solanacearum, Ramlibacter sp., Rhizobium leguminosarum, Rhizobium meliloti, Rhodococcus rhodochrous, Salmonella enteritidis, Salmonella enterica, Serratia marcescens, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Sinorhizobium meliloti, Vibrio alginolyticus, Vibrio anguillarum, Vibrio campbellii, Vibrio cholera, Vibrio fischeri, Vibrio harveyi, Vibrio mimicus, Vibrio natriegens, Vibrio parahaemolyticus, Vibrio proteolytica, Vibrio shiloi, Vibrio vulnificus, Xanthomonas axonopodis, Xanthomonas campestris, Yersinia pestis and Yersinia entomophaga.

[0051] In some examples, a sample of bacteria formulated as a liquid may be dried by any of the techniques described herein. Generally, any type of formulation of the bacteria may be dried and at least some of the viable bacteria in the sample then cultured with the disclosed methods that use humic acid or humic acid related substances. In some examples, the liquid bacterial formulations, or the dried samples, may contain any amount of bacteria. In some examples, the amount of bacteria in the formulations may be from 0.1-99% by weight. In some examples, the amount of bacteria in the formulations may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% by weight.

[0052] In some examples, the liquid bacterial formulations, or the dried samples, may contain 1 x 10¹ to about 1 x 10²⁰ bacteria per milliliter or gram of the formulation. In some examples, about 1 x 10⁴, 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, 1 x 10⁸, 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², 1 x 10¹³, 1 x 10¹⁴, 1 x 10¹⁵, or more bacteria or CFU per milliliter or gram of the formulation may be present.

[0053] In some examples, the liquid bacterial formulations, or the dried samples, may contain non-aqueous carriers. Non-limiting examples of non-aqueous carriers that may be useful in compositions of the present disclosure include disaccharides, maltodextrins, monosaccharides, oligosaccharides, sugar alcohols, peat-based powders and granules, agriculturally acceptable polymers, and the like.

[0054] The non-aqueous carriers may constitute any suitable portion of the liquid bacterial formulations, or the dried samples. In some embodiments, the non-aqueous carrier(s) comprise(s) about 1 to about 99 % (by weight) of the compositions. For example, compositions of the present disclosure, the non-aqueous carrier(s) constitute(s) about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5% or more (by weight) of the composition. In some embodiments, the carrier amount/concentration is about 5 to about 45%, about 10 to about 30%, about 50 to about 99%, about 55% to about 95%, about 60% to about 95%, about 65% to about 90%, about 70 to about 90%, about 75 to about 90%, about 80 to about 90% or about 80 to about 85% (by weight) of the composition.

[0055] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more commercial carriers used in accordance with the manufacturer's recommended amounts/concentrations.

[0056] In some embodiments, the non-aqueous carrier comprises, consists essentially of, or consists of one or more maltodextrins.

[0057] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable maltodextrins, including, but not limited to, maltodextrins having a dextrose equivalent value (DEV) of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, compositions of the present disclosure comprise one or more maltodextrins having a DEV of about 5 to about 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, about 10 to about 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or about 15 to about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the non-aqueous carrier comprises a combination of maltodextrins having a DEV of about 5 to about 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, about 10 to about 11,

12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or about 15 to about 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the non-aqueous carrier comprises one or more maltodextrins having a DEV of about 10 to about 25 (e.g., one or more maltodextrins having a DEV of about 15 to about 20). In some embodiments, the non-aqueous carrier comprises a combination of maltodextrins having a DEV of about 10 to about 25 (e.g., a combination of maltodextrins having a DEV of about 15 to about 20). [0058] Non-limiting examples of maltodextrins that may be useful in compositions of the present disclosure include MALTRIN® Ml 50 (DEV = 15; molecular weight = 1200; Grain Processing Corporation, Muscatine, IA), MALTRIN® Ml 80 (DEV = 18; molecular weight = 1050; Grain Processing Corporation, Muscatine, IA), MALTRIN® M200 (DEV = 20; molecular weight = 900; Grain Processing Corporation, Muscatine, IA), MALTRIN® M250 (DEV = 25; molecular weight = 720; Grain Processing Corporation, Muscatine, IA); MALTRIN QD® M580 (DEV = 16.5-19.9; Grain Processing Corporation, Muscatine, IA); MALTRIN QD® M585 (DEV = 15.0-19.9; Grain Processing Corporation, Muscatine, IA); MALTRIN QD® M600 (DEV = 20.0-23.0; Grain Processing Corporation, Muscatine, IA); GLOBE® Plus 15 DE (Ingredion Inc., Westchester, IL); and combinations thereof. In some embodiments, the maltodextrin (or combination of maltodextrins) has a DEV of 15 to 20.

[0059] Maltodextrins may be incorporated into the liquid bacterial formulations, or the dried samples of the present disclosure, in any suitable amount(s)/concentration(s). In some

embodiments, the maltodextrin(s) comprise about 5 to about 99 % or more (by weight) of the composition. For example, in some embodiments, the non-aqueous carrier comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more of one or more maltodextrins (each and/or collectively) having a DEV value of about 15 to about 20. In some embodiments, the maltodextrin amount/concentration is about 50 to about 95%, about 55% to about 90%, about 60% to about 85%, about 65% to about 80%, or about 70 to about 80% (by weight) of the non-aqueous carrier.

[0060] In some embodiments, the non-aqueous carrier comprises, consists essentially of, or consists of one or more monosaccharides, disaccharides, sugar alcohols and/or oligosaccharides. In some examples, the concentration of these substances in the compositions disclosed herein may be between about 1-90%, 2-80%, 3-70%, 4-60%, 5-50%, 5-40%, 5-30%, 5-20% or 6-15% (by weight).

[0061] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable monosaccharide, including, but limited to, allose, altrose, arabinose, fructose, galactose, glucose, gulose, iodose, lyxose, mannose, ribose, talose, threose and xylose. In some embodiments, the non-aqueous carrier comprises glucose. In some embodiments, the non-aqueous carrier does not comprise glucose.

[0062] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable disaccharide, including, but limited to, cellobiose, chitobiose, gentiobiose, gentiobiulose, isomaltose, kojibiose, lactose, lactulose, laminaribiose, maltose (e.g., maltose monohydrate, anhydrous maltose), maltulose, mannobiose, melibiose, melibiulose, nigerose, palatinose, rutinose, rutinulose, sophorose, sucrose, trehalose, turanose and xylobiose. In some embodiments, the the non-aqueous carrier comprises maltose. In some embodiments, the nonaqueous carrier comprises sucrose. In some embodiments, the non-aqueous carrier comprises trehalose. In some embodiments, the non-aqueous carrier does not comprise trehalose.

[0063] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable oligosaccharide, including, but limited to, fructo-oligosaccharides, galacto-oligosaccharides, mannon-oligosaccharides and raffinose.

[0064] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable sugar alcohol, non-limiting examples of which include sorbitol, xylitol, glycerol, erythritol, threitol, arabitol, ribitol, mannitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetraitol and polyglycitol.

[0065] Mono-, di- and oligosaccharides may be incorporated into the liquid bacterial formulations, or the dried samples of the present disclosure, in any suitable

amount(s)/concentration(s). In some embodiments, the mono-, di- and/or oligosaccharide(s) comprise(s) about 5 to about 95% (by weight) of the composition. For example, in some embodiments, the non-aqueous carrier comprises about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more (by weight) of one or more mono-, di- and/or oligosaccharides. In some embodiments, the mono-, di- and/or oligosaccharide amount/concentration is about 1 to about 65%, about 5% to about 20%, about 10% to about 25%, about 20% to about 50%, or about 30 to about 60% (by weight) of the nonaqueous carrier. [0066] In some embodiments, the non-aqueous carrier comprises, consists essentially of, or consists of one or more malt extracts.

[0067] In some embodiments, the non-aqueous carrier comprises, consists essentially of, or consists of one or more peat-based powders and/or granules.

[0068] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable peat-based powder(s) and/or granule(s).

[0069] Peat-based powders and/or granules may be incorporated into the compositions of the present disclosure in any suitable form.

[0070] Peat-based powders and/or granules may be incorporated into the liquid bacterial formulations, or the dried samples of the present disclosure in any suitable

amount(s)/concentration(s). In some embodiments, the peat-based powder(s) and/or granuale(s) comprise(s) about 5 to about 95 % (by weight) of the composition. For example, in some embodiments, the non-aqueous carrier comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more (by weight) of one or more peat-based powders and/or granuales. In some embodiments, the peat extract amount/concentration is about 50 to about 99%, about 55% to about 95%, about 60% to about 90%, about 65% to about 90%, or about 70 to about 90% (by weight) of the non-aqueous carrier.

[0071] In some embodiments, the non-aqueous carrier comprises, consists essentially of, or consists of one or more agriculturally acceptable polymers.

[0072] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable agriculturally acceptable polymer(s), including, but not limited to, biodegradable polymers and synthetic polymers. For example, in some embodiments, compositions of the present disclosure comprise agar, alginate, carrageenan, cellulose, guar gum, locust bean gum, methylcellulose, pectin, polycaprolactone, polylactide, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, starch and xanthan gum. [0073] Non-limiting examples of polymers that may be useful in compositions of the present disclosure include TICAXAN® xanthan powders, such as PRE-HYDRATED® TICAXAN® Rapid-3 Powder (TIC Gums, White Marsh, MD) and combinations thereof.

[0074] Additional examples of polymers that may be included in the compositions of the present disclosure may be found in Pouci, et al. AM. J. AGRIC. BIOL. SCI. 3(1):299 (2008).

[0075] Agriculturally acceptable polymers may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). In some embodiments, the agriculturally acceptable polymer(s) comprise(s) about 5 to about 95 % (by weight) of the composition. For example, in some embodiments, the non-aqueous carrier comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more (by weight) of one or more agriculturally acceptable polymers. In some embodiments, the agriculturally acceptable polymers amount/concentration is about 50 to about 99%, about 55% to about 95%, about 60% to about 90%), about 65%> to about 90%>, or about 70 to about 90%> (by weight) of the nonaqueous carrier.

[0076] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more commercial polymers used in accordance with the manufacturer's recommended amounts/concentrations.

[0077] It is to be understood that monosaccharides, disaccharides, oligosaccharides, maltodextrins, malt extracts, agriculturally acceptable polymers and peat-based powders/granules may be combined to form dry non-aqueous carriers having beneficial properties. Thus, in some embodiments, compositions of the present disclosure comprise a dry non-aqueous carrier that comprises one or more maltodextrins in combination with one or more mono-, di- and/or oligosaccharides, one or more sugar alcohols, one or more malt extracts, one or more

agriculturally acceptable polymers and/or one or more peat-based powders and/or granules.

[0078] Maltodextins and mono-, di- and/or oligosaccharides may be incorporated into compositions of the present disclosure in any suitable ratio(s). In some embodiments, the nonaqueous carrier has a maltodextrin:(mono-, di- and/or oligosaccharide) (e.g.,

maltodextrin: maltose) ratio of about 1 :99 to about 99: 1 (by weight, based upon the respective weight percentages of the maltodextrin(s) and mono-, di- and/or oligosaccharide(s) in the nonaqueous carrier). For example, in some embodiments, the non-aqueous carrier has a

maltodextrin:(mono-, di- and/or oligosaccharide) (e.g., maltodextrin: maltose) ratio of about 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 67:33, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5 or more. In some embodiments, the maltodextrin: (mono-, di- and/or oligosaccharide) (e.g., maltodextrimmaltose) ratio is about 45:55 to about 95:5. Thus, in some embodiments, the compositions of the present disclosure comprise one or more

maltodextrins having a DEV of about 15 to about 20 and one or more mono-, di- and/or oligosaccharides (e.g., maltose) in a ratio of about 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5.

[0079] Maltodextins and malt extracts may be incorporated into the liquid bacterial formulations, or the dried samples of the present disclosure, in any suitable ratio(s). In some embodiments, the non-aqueous carrier has a maltodextrin: malt extract ratio of about 1 :99 to about 99: 1 (by weight, based upon the respective weight percentages of the maltodextrin(s) and malt extract(s) in the non-aqueous carrier). For example, in some embodiments, the non-aqueous carrier has a maltodextrin: malt extract ratio of about 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5 or more. In some embodiments, the maltodextrin: malt extract ratio is about 45:55 to about 95:5. Thus, in some embodiments, the compositions of the present disclosure comprise one or more

maltodextrins having a DEV of about 15 to about 20 and one or more malt extracts in a ratio of about 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5.

[0080] Maltodextins and peat-based powders and/or granules may be incorporated into the liquid bacterial formulations, or the dried samples of the present disclosure, in any suitable ratio(s). In some embodiments, the non-aqueous carrier has a maltodextrin: peat powder/granuale ratio of about 1 : 99 to about 99: 1 (by weight, based upon the respective weight percentages of the maltodextrin(s) and peat powder(s)/granuale(s) in the non-aqueous carrier). For example, in some embodiments, the non-aqueous carrier has a maltodextrin: peat powder/granuale ratio of about 5:95, 10:90, 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5 or more. In some embodiments, the maltodextrin: peat powder/granuale ratio is about 45:55 to about 95:5. Thus, in some embodiments, the compositions of the present disclosure comprise one or more maltodextrins having a DEV of about 15 to about 20 and one or more peat-based powders and/or granuales in a ratio of about 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85: 15, 90: 10, 95:5.

[0081] Non-aqueous carriers may be incorporated into the liquid bacterial formulations, or the dried samples of the present disclosure, in any suitable amount(s)/concentration(s).

[0082] In some embodiments, the non-aqueous carrier(s) comprise(s) about 5 to about 99.9% (by weight) of the composition. For example, in some embodiments, the non-aqueous carrier(s) constitute(s) about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5%) or more (by weight) of the composition. In some embodiments, the non-aqueous carrier amount/concentration is about 50 to about 99%, about 55% to about 95%, about 60%> to about 95%, about 65% to about 90%, about 70 to about 90%, about 75 to about 90%, about 80 to about 90%) or about 80 to about 85%> (by weight) of the composition.

[0083] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more commercial carriers used in accordance with the manufacturer's recommended amounts/concentrations.

[0084] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable agriculturally acceptable dispersant(s), including, but not limited to, surfactants and wetting agents.

[0085] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more anionic surfactants. For example, in some

embodiments, the compositions of the present disclosure comprise one or more water-soluble anionic surfactants and/or one or more water-insoluble anionic surfactants, optionally one or more anionic surfactants selected from the group consisting of alkyl carboxylates (e.g., sodium stearate), alkyl sulfates (e.g., alkyl lauryl sulfate, sodium lauryl sulfate), alkyl ether sulfates, alkyl amido ether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates, alkyl aryl sulfonates, alkyl sulfonates, alkyl amide sulfonates, alkyl aryl sulfonates, alkyl benzene sulfonates, alkyl diphenyloxide sulfonate, alpha-olefin sulfonates, alkyl naphthalene sulfonates, paraffin sulfonates, alkyl sulfosuccinates, alkyl ether sulfosuccinates, alkylamide sulfosuccinates, alkyl sulfosuccinamates, alkyl sulfoacetates, alkyl phosphates, alkyl ether phosphates, acyl sarconsinates, acyl isethionates, N-acyl taurates, N-acyl-N-alkyltaurates, benzene sulfonates, cumene sulfonates, dioctyl sodium sulfosuccinate, ethoxylated sulfosuccinates, lignin sulfonates, linear alkylbenzene sulfonates, monoglyceride sulfates, perfluorobutanesulfonate,

perfluorooctanesulfonate, phosphate ester, styrene acrylic polymers, toluene sulfonates and xylene sulfonates.

[0086] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more cationic surfactants. For example, in some

embodiments, the compositions of the present disclosure comprise one or more pH-dependent amines and/or one or more quaternary ammonium cations, optionally one or more cationic surfactants selected from the group consisting of alkyl trimethyl ammonium salts (e.g., cetyl trimethylammonium bromide, cetyl trimethylammonium chloride), cetylpyridinium chloride, benzalkonium chloride, benzethonium chloride, 5-Bromo-5-nitro-l,3-dioxane,

dimethyldioctadecylammonium chloride, cetrimonium bromide, dioctadecyldimethylammonium bromide and/or octenidine dihydrochloride.

[0087] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more nonionic surfactants. For example, in some

embodiments, compositions of the present disclosure comprise one or more water-soluble nonionic surfactants and/or one or more water-insoluble nonionic surfactants, optionally one or more nonionic surfactants selected from the group consisting of alcohol ethoxylates (e.g., TERGITOL™ 15-S surfactants, such as TERGITOL™15-S-9 (The Dow Chemical

Company,Midland, MI)), alkanolamides, alkanolamine condensates, carboxylic acid esters, cetostearyl alcohol, cetyl alcohol, cocamide DEA, dodecyldimethylamine oxides, ethanolamides, ethoxylates of glycerol ester and glycol esters, ethylene oxide polymers, ethylene oxide- propylene oxide copolymers, glucoside alkyl ethers, glycerol alkyl ethers (e.g., ), glycerol esters, glycol alkyl ethers (e.g., polyoxyethylene glycol alkyl ethers, polyoxypropylene glycol alkyl ethers,), glycol alkylphenol ethers (e.g., polyoxyethylene glycol alkylphenol ethers,), glycol esters, monolaurin, pentaethylene glycol monododecyl ethers, poloxamer, polyamines, polyglycerol polyricinoleate, polysorbate, polyoxyethylenated fatty acids, polyoxyethylenated mercaptans, polyoxyethylenated polyoxyproylene glycols, polyoxyethylene glycol sorbitan alkyl esters, polyethylene glycol-polypropylene glycol copolymers, polyoxyethylene glycol octylphenol ethers, polyvinyl pynolidones, sugar-based alkyl polyglycosides, sulfoanylamides, sorbitan fatty acid alcohol ethoxylates, sorbitan fatty acid ester ethoxylates, sorbitan fatty acid ester and/or tertiary acetylenic glycols.

[0088] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise at least one nonionic surfactant. In some embodiments, the compositions of the present disclosure comprise at least one water insoluble nonionic surfactant and at least one water soluble nonionic surfactant. In some embodiments, the compositions of the present disclosure comprise a combination of nonionic surfactants having hydrocarbon chains of substantially the same length.

[0089] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more zwitterionic surfactants. For example, in some embodiments, the compositions of the present disclosure comprise one or more betaines and/or one or more sultaines, optionally one or more zwitterionic surfactants selected from the group consisting of 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine and/or one or more sphingomyelins.

[0090] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more soaps and/or organosilicone surfactants. For example, in some embodiments, the compositions of the present disclosure comprise one or more alkali metal salts of fatty acids.

[0091] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more wetting agents. For example, in some embodiments, the compositions of the present disclosure comprise one or more naphthalene sulfonates, optionally one or more alkyl naphthalene sulfonates (e.g., sodium alkyl naphthalene sulfonate), one or more isopropyl naphthalene sulfonates (e.g., sodium isopropyl naphthalene sulfonate) and/or one or more butyl naphthalene sulfonates (e.g., sodium n-butyl naphthalene sulfonate). [0092] Selection of appropriate surfactants will depend on the intended application(s) and the microorganism(s) present in the composition. In general, the surfactant(s) will have low toxicity for the bacteria in the composition and for the plant part(s) to which the composition is to be applied. In some embodiments, the surfactant(s) will be selected to wet and/or emulsify one or more soils.

[0093] Non-limiting examples of dispersants that may be useful in compositions of the present disclosure include Atlox™ (e.g., 4916, 4991; Croda International PLC, Edison, NJ), BIO-SOFT® (e.g., N series, such as Nl-3, Nl-7, Nl-5, Nl-9, N23-3, N2.3-6.5, N25-3, N25-7, N25-9, N91-2.5, N91-6, N91-8; Stepan Company, Northfield, IL), MAKON® nonionic surfactants (e.g., DA-4, DA-6 and DA-9; Stepan Company, Northfield, IL), MORWET® powders (Akzo Nobel Surface Chemistry LLC, Chicago, IL), MULTIWET™ surfactants (e.g., MO-70R, MO-85P, MO-85P-PW-(AP); Croda International PLC, Edison, NJ), SILWET® L-77 (Helena Chemical Company, Collierville, TN), SPAN™ surfactants (e.g., 20, 40, 60, 65, 80 and 85; Croda Inc., Edison NJ), TAMOL™ dispersants (The Dow Chemical Company,Midland, MI ), TERGITOL™ surfactants (e.g., 15-S-9, TMN-6, TMN-100X and XD; The Dow Chemical Company,Midland, MI), TERSPERSE surfactants (e.g., 2001, 2020, 2100, 2105, 2158, 2700, 4894 and 4896; Hunstman Corp., The Woodlands, TX), TRITON™ surfactants (e.g., X-100; The Dow Chemical Company ,Midland, MI), TWEEN® surfactants (e.g., TWEEN® 20, 21, 22, 23, 28, 40, 60, 61, 65, 80, 81 and 85; Croda International PLC, Edison, NJ) and combinations thereof.

[0094] Dispersants may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). In some embodiments, the dispersant(s) comprise(s) about 0.1 to about 25 % (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25% or more (by weight) of one or more dispersants. In some embodiments, the dispersant (s) comprise(s) about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 to about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 % (by weight) of the composition. [0095] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more commercial wetting agents and/or one or more surfactants used in accordance with the manufacturer's recommended amounts/concentrations.

[0096] Additional examples of dispersants that may be included in the compositions of the present disclosure may be found in BAIRD & ZUBLENA. 1993. SOIL FACTS: USING WETTING AGENTS (NONIONIC SURFACTANTS) ON SOIL (North Carolina Cooperative Extension Service Publication AG-439-25) (1993); BURGES, FORMULATION OF MICROBIAL BIOPESTICIDES:

BENEFICIAL MICROORGANISMS, NEMATODES AND SEED TREATMENTS (Springer Science & Business Media) (2012); MCCARTY, WETTING AGENTS (Clemson University Cooperative Extension Service Publication) (2001).

[0097] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable excipient(s), including, but not limited to, anti-freezing agents, drying agents, safeners and pH buffers.

[0098] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable agriculturally acceptable anti-freezing agent(s), including, but not limited to, ethylene glycol, glycerin, propylene glycol and urea.

[0099] In some embodiments, the compositions of the present disclosure comprise one or more commercial anti-freezing agents used in accordance with the manufacturer's recommended amounts/concentrations.

[00100] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable agriculturally acceptable drying agent(s), including, but not limited to, drying powders. For example, in some embodiments, the compositions of the present disclosure comprise calcium stearate, clay (e.g., attapulgite clay, montmorillonite clay), graphite, magnesium stearate, magnesium sulfate, powdered milk, silica (e.g., fumed silica,

hydrophobically-coated silica, precipitated silica), soy lecithin and/or talc.

[00101] Non-limiting examples of drying agents that may be useful in compositions of the present disclosure include AEROSIL® hydrophobic fumed silica powders (Evonik Corporation, Parsippany, NJ), BENTOLITE® powders (BYK-Chemie GmbH, Wesel, Germany), SIPERNAT® silica powders (Evonik Corporation, Parsippany, NJ) and combinations thereof.

[00102] Additional examples of drying agents that may be included in the compositions of the present disclosure may be found in BURGES, FORMULATION OF MICROBIAL BIOPESTICIDES:

BENEFICIAL MICROORGANISMS, NEMATODES AND SEED TREATMENTS (Springer Science & Business Media) (2012).

[00103] Drying agents may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). In some embodiments, the compositions of the present disclosure comprise about 0.5 to about 10 grams of drying powder per liter of composition. For example, the compositions of the present disclosure may comprise about 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 grams or more of drying powder per liter of the composition. In some embodiments, the amount/concentration of drying agent(s) comprise(s) calcium stearate, attapulgite clay, montmorillonite clay, graphite, magnesium stearate, silica (e.g., fumed silica, hydrophobically- coated silica and/or precipitated silica) and/or talc.

[00104] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more commercial drying agents used in accordance with the manufacturer's recommended amounts/concentrations.

[00105] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable agriculturally acceptable dust suppressant(s), including, but not limited to, adhesives, glycerin, mineral oils, paraffinic oils, vegetable oils and synthetic polymers. It is to be understood that some compounds may act as both an adhesive and a dust suppressant. Indeed, the dust suppressant activity of many compounds arises from their ability to adhere dust particles to other heavier particles. For example, in some embodiments of the present disclosure, one or more oils is used to adhere microbial spores in the composition to larger particles comprising one or more maltodextrins.

[00106] Non-limiting examples of dust suppressants that may be useful in compositions of the present disclosure include ARENAPRO, BIORAIN and ROADKILL (Dustkill LLC, Columbus, IN), BIO-SOFT® surfactants (e.g., N series, such as Nl-3, Nl-7, Nl-5, Nl-9, N23-3, N2.3-6.5, N25-3, N25-7, N25-9, N91-2.5, N91-6, N91-8; Stepan Company, Northfield, IL), DURASOIL (Soilworks, LLC, Scottsdale, AZ), DUSGON (Dupont (Australia) Ltd, Macquarie Park,

Australia), DUSTRX (Seaco Technologies, Inc., Bakersfield, CA), SUN AG® oils

(HollyFrontier Refining & Marketing, LLC, Plymouth Meeting, PA), SUNSPRAY® oils (e.g., 6N, 6E, 8N, 1 IN; HollyFrontier Refining & Marketing, LLC, Plymouth Meeting, PA),

SUNSPRAY® ULTRA-FINE® oils (HollyFrontier Refining & Marketing, LLC, Plymouth Meeting, PA) , TOMADOL® surfactants (e.g., 23-1, 23-3, 23-5, 23-6.5; Air Products and Chemicals, Inc., Allentown, PA) and combinations thereof.

[00107] Dust suppressants may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s).

[00108] In some embodiments, dust suppressants comprise about 0.1 to about 30% (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 6, 7, 8, 9, 10% or more (by weight) of one or more dust suppressants. In some embodiments, the dust suppressant amount/concentration is about 1 to about 2, 3, 4, 5, 6, 7, 8, 9 or 10%, about 1.5 to about 2, 3, 4, 5, 6, 7, 8, 9 or 10%, about 2 to about 3, 4, 5, 6, 7, 8, 9 or 10%, about 2.5 to about 3, 4, 5, 6, 7, 8, 9 or 10% or about 3 to about 4, 5, 6, 7, 8, 9 or 10%) (by weight) of the inculant composition.

[00109] It is to be understood that some compounds may act as both a dispersant and a dust suppressant. For example, in some embodiments, the compositions of the present disclosure comprise one or more surfactants, such as BIO-SOFT® N23-3, that disperse microorganisms within the composition and suppress dusting.

[00110] As noted above, the non-aqueous carrier in the compositions of the present invention may act as a protectant. It is to understood that the compositions of the present disclosure may comprise additional protectants.

[00111] Protectants may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). [00112] In some embodiments, the protectant(s) comprise(s) about 0.1 to about 25 % (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25% or more (by weight) of one or more protectants. In some embodiments, the protectant amount/concentration is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 to about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 % (by weight) of the

composition.

[00113] In some embodiments, the protectant amount/concentration is effective to ensure that at least about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65% or more of the microbial spores/vegetative cells in the composition survive following storage at 10,

15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85% or more relative humidity for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104 weeks or more.

[00114] In some embodiments, the protectant amount/concentration is effective to ensure that at least about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65% or more of the microbial spores/vegetative cells in the composition survive following desiccation (of about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more) and storage at 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85% or more relative humidity for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104 weeks or more.

[00115] In some embodiments, the protectant amount/concentration is effective to ensure that at least about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65% or more of the microbial spores/vegetative cells in the composition survive following

cryopreservation at or below -80°C for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 20, 21, 22, 23, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104 weeks or more.

[00116] In some embodiments, the compositions of the present disclosure compri

more sugar alcohols (e.g., sorbitol). [00117] Sugar alcohols may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). In some embodiments, the sugar alcohol(s)

comprise(s)about 0.005 to about 10 % (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5% or more (by weight) of one or more sugar alcohols (e.g., sorbitol).

[00118] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more peat extracts.

[00119] Peat extracts may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). In some embodiments, the peat extract(s) comprise(s) about 0.1 to about 25 % (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25% or more (by weight) of one or more peat extracts. In some embodiments, the peat extract amount/concentration is about 0.5 to about 10% (by weight) of the the composition.

[00120] In some embodiments, the compositions of the present disclosure comprise one or more peptones.

[00121] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure comprise one or more skim milk extracts.

[00122] Skim milk extracts may be incorporated into the compositions of the present disclosure in any suitable amount/concentration. In some embodiments, skim milk extracts comprise about 0.1 to about 25 %> (by weight) of the the composition. For example, the compositions of the present disclosure may comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25% or more (by weight) of skim milk extracts. In some embodiments, the skim milk extract amount/concentration is about 0.5 to about 10%) (by weight) of the composition.

[00123] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more hygroscopic polymers. For example, the compositions of the present disclosure may comprise one or more albumins, alginates, celluloses, gums (e.g., cellulose gum, guar gum, gum arabic, gum combretum, xantham gum), methyl celluloses, nylons, pectins, polyacrylic acids, polycarbonates, polyethylene glycols (PEG),

polyethylenimines (PEI), polylactides, polymethylacrylates (PMA), polyurethanes, polyvinyl alcohols (PVA), polyvinylpyrrolidones (PVP), propylene glycols, sodium carboxymethyl celluloses and/or starches.

[00124] Non-limiting examples of hygroscopic polymers that may be useful in compositions of the present disclosure include AGRIMER™ polymers (e.g., 30, AL-10 LC, AL-22, AT/ATF, VA 3E, VA 31, VA 5E, VA 51, VA 6, VA 6E, VA 7E, VA 71, VEMA AN-216, VEMA AN-990, VEMA AN-1200, VEMA AN-1980, VEMA H-815MS; Ashland Specialty Ingredients,

Wilmington, DE), EASYSPERSE™ polymers (Ashland Specialty Ingredients, Wilmington, DE); DISCO™ AG polymers (e.g., L-250, L-280, L-285, L-286, L-320, L-323, L-517, L-519, L- 520, L800; Incotec Inc., Salinas, CA), KELZAN® polymers (Bri-Chem Supply Ltd., Calgary, Alberta, CA), SEEDWORX™ polymers (e.g., Bio 200; Aginnovation, LLC, Walnut Groove, CA), TABULOSE® gels (e.g., SC-580, SC-612, SC-613, SC-681; Blanver Farmoquimica, Boca Raton, FL), TICAXAN® xanthan powders (TIC Gums, White Marsh, MD) and combinations thereof.

[00125] Additional examples of hygroscopic polymers that may be included in the

compositions of the present disclosure may be found in Pouci, et al. AM. J. AGRIC. BIOL. SCI. 3(1):299 (2008).

[00126] Hygroscopic polymers may be incorporated into the liquid bacterial formulations, or the dried samples of the present disclosure in any suitable amount(s)/concentration(s). In some embodiments, the hygroscopic polymer(s) comprise(s) about 0.1 to about 25 % (by weight) of the composition. In some embodiments, the hygroscopic polymer(s) comprise(s) about 0.5 to about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 % (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 25% or more (by weight) of one or more hygroscopic polymers. In some embodiments, the hygroscopic polymer amount/concentration is about 0.5 to about 10% (by weight) of the composition. In some embodiments, the hygroscopic polymer amount/concentration is about 0.5 to about 5% (by weight) of the composition.

[00127] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure, comprise one or more commercial hygroscopic polymers used in accordance with the manufacturer's recommended amounts/concentrations.

[00128] In some embodiments, the liquid bacterial formulations, or the dried samples of the present disclosure comprise one or more oxidation control components.

[00129] The compositions of the present disclosure may comprise any suitable oxidation control component(s), including, but not limited to, antioxidants and/or oxygen scavengers. In some embodiments, the oxidation control component is/comprises ascorbic acid and/or glutathione.

[00130] In some embodiments, the liquid bacterial formulations, or the dried samples, comprise one or more antioxidants. For example, in some embodiments, the compositions of the present disclosure comprise ascorbic acid, ascorbyl palmitate, ascorbyl stearate, calcium ascorbate, carotenoids, lipoic acid, phenolic compounds (e.g., flavonoids, flavones, flavonols), potassium ascorbate, sodium ascorbate, thiols (e.g., glutathione, lipoic acid, N-acetyl cysteine), tocopherols, tocotrienols, ubiquinone and/or uric acid.

[00131] Non-limiting examples of antioxidants that may be useful in compositions of the present disclosure include those that are soluble in the cell membrane (e.g., alpha tocopherol (vitamin E), ascorbyl palmitate), those that are soluble in alcohols (e.g., IRGANOX®

antioxidants (BASF Schweiz AG, Basel, Switzerland)) and those that are soluble in water (e.g., ascorbic acid and isomers or ascorbic acid, sodium or potassium salts of ascorbic acid or isomers or ascorbic acid, glutathione, sodium or potassium salts of glutathione). In some embodiments, use of a membrane-soluble antioxidant necessitates the addition of one or more surfactants to adequately disperse the antioxidant within the composition.

[00132] In some embodiments, the compositions of the present disclosure comprise one or more commercial antioxidants used in accordance with the manufacturer's recommended amounts/concentrations. [00133] In some embodiments, the liquid bacterial formulations, or the dried samples, comprise one or more naturally occurring or synthetic oxygen scavengers. For example, in some embodiments, the compositions of the present disclosure comprise ascorbic acid, ascorbate salts, catechol and/or sodium hydrogen carbonate.

[00134] In some embodiments, the compositions of the present disclosure comprise one or more commercial oxygen scavengers used in accordance with the manufacturer's recommended amounts/concentrations.

[00135] Oxidation control components may be incorporated into the liquid bacterial formulations, or the dried samples compositions of the present disclosure in any suitable amount(s)/concentration(s).

[00136] In some embodiments, the oxidation control component(s) comprise(s) about 0.0001 to about 5 % or more (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5% or more of one or more oxidation control components. In some embodiments, the amount/concentration of oxidation control components is about 0.005 to about 2% (by weight) of the composition. In some embodiments, the oxidation control component(s) is/are present in a concentration ranging from about 1 x 10^"20 M to about 1 x 10^"1 M. For example, one or more oxidation control components may be added at a concentration of 1 x 10^"20 M, 1 x 10^"19 M, 1 x 10^"18 M, 1 x 10^"17 M, 1 x 10^"16 M, 1 x 10^"15 M, 1 x 10^"14 M, 1 x 10^"13 M, 1 x 10^"12 M, 1 x 10^"11 M, 1 x 10^"10 M, 1 x 10^"9 M, 1 x 10^"8 M, 1 x 10^"7 M, 1 x 10^"6 M, 1 x 10^"5 M, 1 x 10^"4 M, 1 x 10^"3 M, 1 x 10^"2 M, 1 x 10^"1 M or more.

[00137] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable safener(s), including, but not limited to, napthalic anhydride.

[00138] Safeners may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). [00139] In some embodiments, the compositions of the present disclosure comprise one or more commercial safeners used in accordance with the manufacturer's recommended

amounts/concentrations.

[00140] The liquid bacterial formulations, or the dried samples compositions of the present disclosure may comprise any suitable pH buffer(s), including, but not limited to, potassium phosphate monobasic and potassium phosphate dibasic.

[00141] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable adhesive(s), including, but not limited to, adhesive compositions comprising one or more maltodextrins and/or one or more mono-, di- or oligosaccharides. The compositions of the present disclosure may be formulated into any suitable type of composition, including, but not limited to, seed coatings, soil formulations and foliar formulations.

[00142] The liquid bacterial formulations, or the dried samples of the present disclosure, may comprise any suitable anti-settling agent(s), including, but not limited to, polyvinyl acetate, polyvinyl alcohols with different degrees of hydrolysis, polyvinylpyrrolidones, polyacrylates, acrylate-, polyol- or polyester-based paint system binders which are soluble or dispersible in water, moreover copolymers of two or more monomers such as acrylic acid, methacrylic acid, itaconic acid, maleic acid, fumaric acid, maleic anhydride, vinylpyrrolidone, ethylenically unsaturated monomers such as ethylene, butadiene, isoprene, chloroprene, styrene,

divinylbenzene, ot-methylstyrene or p-m ethyl styrene, further vinyl halides such as vinyl chloride and vinylidene chloride, additionally vinyl esters such as vinyl acetate, vinyl propionate or vinyl stearate, moreover vinyl methyl ketone or esters of acrylic acid or methacrylic acid with monohydric alcohols or polyols such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethylene methacrylate, lauryl acrylate, lauryl methacrylate, decyl acrylate, N,N-dimethylamino- ethyl methacrylate, 2-hydroxyethyl methacrylate, 2-hydroxypropyl methacrylate or glycidyl methacrylate, furthermore diethyl esters or monoesters of unsaturated dicarboxylic acids, furthermore (meth)acrylamido-N-methylol methyl ether, amides or nitriles such as acrylamide, methacrylamide, N-methylol(meth)acrylamide, acrylonitrile, methacrylonitrile, and also N- substituted maleiraides and ethers such as vinyl butyl ether, vinyl isobutyl ether or vinyl phenyl ether, and combinations thereof. [00143] Anti-settling agents may be incorporated into the compositions of the present disclosure in any suitable amount(s)/concentration(s). In some embodiments, the compositions of the present disclosure comprise about 0.0001 to about 10% or more (by weight) of the composition. For example, the compositions of the present disclosure may comprise about 0.0005, 0.001, 0.002, 0.003, 0.004, 0.005, 0.0075, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.75, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10% or more of one or more anti-settling agents. In some embodiments, the amount/concentration of anti-settling agents is about 0.01 to about 5% (by weight) of the composition.

[00144] In some embodiments, the compositions of the present disclosure comprise one or more commercial anti-settling agents used in accordance with the manufacturer's recommended amounts/concentrations.

[00145] The liquid bacterial formulations, or the dried samples compositions of the present disclosure, may comprise any suitable biostimulant(s), including, but not limited to, seaweed extracts (e.g., Ascophyllum nodosum extracts, such as alginate, Ecklonia maxima extracts, etc.), humic acids (e.g., potassium humate), fulvic acids, myo-inositol, glycine and combinations thereof.

Humic acids, salts, and analogs

[00146] Soil organic matter may be classified as a humic substance or a non-humic substance. Humic substances are composed of altered or transformed components of plants, animals, microbes, and the like (e.g., decomposed organic matter). Non-humic substances include unaltered remains (e.g., not decomposed) of plants, animals, microbes, and the like. Humic substances are generally thought to include a humic acid component, a fulvic acid component, and a humin component. The humic acid component, and substances that may contain all or part of the humic acid component, is disclosed herein as capable of increasing the efficiency of plating of unculturable bacteria from samples.

[00147] These three components of humic substances - humic acid, fulvic acid, and humin - are defined, in part, based on their aqueous solubilities at different pH values. The humic acid component, for example, is generally water soluble at alkaline pH, but becomes less soluble under acidic conditions. In some examples, humic acid may be defined as the fraction of humic substances that are water insoluble at pH 2, but are increasingly soluble at higher pH values. The fulvic acid component is generally soluble in water at all pH values. The humin component is generally insoluble at all pH values.

[00148] Chemically, humic acid is a complex mixture of weak aliphatic and aromatic organic acids, often containing phenolic and carboxylic substituents. Humic acids may be called polydisperse because of their variable chemical features. The molecular sizes of humic acids may range, in some examples, from approximately about 10,000 to about 100,000 daltons.

Humic acids may readily form salts with inorganic trace mineral elements. Both humic acids and salts thereof can be used and may be active in the methods disclosed herein.

[00149] Humic substances, and therefore humic acid, may be components of soil (e.g., humus), peat, lignite, coal, lake and stream sediments, seawater, and shale (e.g., Leonardite). Humic acid may be obtained or extracted from certain of these substances (e.g., convenient sources may be humus rich soil, peat moss, compost) using various methods. Humic acid may also be obtained from systems set up to facilitate degradation of organic materials (e.g., plant material) so that humic acid is produced. Humic acid may also be formed by polymerization of substances like polyphenols. Some of these methods are described in, for example, US Patent No. 5,854,032. Other methods for extracting or producing humic acids may be used. Humic acids can also be purchased commercially (e.g., Sigma- Aldrich No. 53680; Alfa Aesar No. 41747). The above-mentioned substances - like peat, lignite, coal, sediments, seawater, shale, and the like - are also within the scope of materials that increase plating efficiency of unculturable bacteria.

[00150] Salts of humic acid are within the scope of materials that can increase the efficiency of plating or recovery of unculturable bacteria from samples. In some examples, formation of salts of humic acid depends on the ability of carboxyl and/or hydroxyl groups therein to dissociate their hydrogen ions and bind to positive cations (e.g., metal cations like iron, copper, zinc, calcium, manganese, magnesium, and the like). Salts of humic acid can be purchased commercially (Sigma-Aldrich No. H16752). [00151] Humic acid analogs and synthetic humic acids (a humic acid analog may also be synthetic) also exist and are within the scope of materials that may increase the efficiency of plating of unculturable bacteria. In some examples, certain quinones, one being anthraquinone- 2, 6-disulfonate (AQDS), are considered analogs of humic acid. Synthetic humic acids can be made by methods known in the art (e.g., V. A. Litvin, R. L. Galagan. "Synthesis and Properties of Synthetic Analogs of Natural Humic Acids." Russian Journal of Applied Chemistry 85, no. 2, 2012).

[00152] Humic acid may be fractionated and some of the fractions may be successfully used in the methods disclosed herein. In some examples of fractionating, humic acid is added to an aqueous solution of 0.1 M ammonium bicarbonate at a slightly basic pH. Insoluble material is removed from the mixture. The remaining solution is passed through a filter that retains molecules larger than 5,000 molecular weight on the filter, while molecules smaller than 5,000 molecular weight pass through the filter. The material retained on the filter may be shown to possess the activity of increasing the efficiency of plating of unculturable bacteria. Other methods of fractionating humic acid may be used.

[00153] Herein, humic acid, salts thereof, analogs thereof, and peat, may include leonardite humic acids, lignite humic acids, peat humic acids or water-extracted humic acids. In some examples, humic acid, salts thereof, analogs thereof, and peat, may include ammonium humate, boron humate, potassium humate and/or sodium humate. In some examples, one or more of ammonium humate, boron humate, potassium humate and sodium humate is/are excluded.

Nonlimiting examples of humic acids that may be useful various examples may include MDL Number MFCD00147177 (CAS Number 1415-93-6), MDL Number MFCD00135560 (CAS Number 68131-04-4), MDL Number MFCS22495372 (CAS Number 68514-28-3), CAS Number 93924-35-7, and CAS Number 308067-45-0.

Recovery of dried bacteria

[00154] Dried bacterial populations likely contain viable but unculturable bacteria.

Unculturable bacteria were generally once culturable under a specific set of conditions, but became unculturable at a later time under those same culture conditions. In some examples, application of stress (e.g., drying) to the culturable bacteria results in the bacteria becoming unculturable. Recovery of unculturable bacteria may occur when the unculturable bacteria become culturable later. In some examples, it may be possible to treat or stimulate the bacteria to regain culturability under the specific conditions under which they were previously

unculturable. In some examples, it may be possible to culture the unculturable bacteria by changing the specific culture conditions. Herein, are disclosed methods for culturing dried bacteria by changing the conditions - by adding to the bacterial culture medium, humic acid, salts thereof, analogs thereof, or peat. In some examples, humic acid may be added without salts, analogs, or peat. In some examples, salts of humic acid may be added, without humic acid, analogs, or peat. In some examples, analogs of humic acid may be added, without humic acid, salts thereof, or peat. In some examples, peat may be added, without humic acid, salts thereof, or analogs thereof.

[00155] In some examples, the humic acids and/or related substances may be added to any bacterial medium. In some examples, the growth media may include YEM, R2A, TSA, LB, NA, ISP2, Jensen's, and the like. Media used for culturing bacteria may be liquid, semisolid or solid. Semisolid or solid medium may be made, in some examples, by adding a gelling agent to a liquid medium. A common gelling agent is agar. However, a number of other gelling agents exist and may be used. Examples include agarose, alginic acid, carrageenan, gelatin, gellan gum, guar gum, xanthan gum, and others. Generally, bacteria plated on a semisolid or solid medium may divide and form colonies after a time when the medium is placed in an environment conducive to growth of bacteria (e.g., 2-5 days of incubation at 30°C in an ambient atmosphere). However, these conditions (e.g., days of incubation, temperature, atmosphere) may vary and optimal conditions may be empirically determined.

[00156] Different forms of humic acid may require different concentrations within media to produce increased efficiency of plating of bacteria isolated from samples, as compared to media that lacks the humic acids (e.g., the optimal concentration of humic acid may not be the same as the optimal concentration of a salt of humic acid). In some examples, concentrations of any of the various humic acid forms above 0% (weight/volume) may be used. In some examples, humic acid forms may be used at concentrations above 0% and less than about 5% (e.g., 0.25, 0.50, 1.50, 2.00, 2.50%). In some examples, humic acid forms may be used at concentrations above 0% and less than about 0.25% (e.g., 0.10, 0.15, 0.20, 0.25%). In some examples, a concentration of humic acid used in the medium is not 0.1% or is above 0.1%. In some examples, concentrations of humic acid between about 0-5% or 0.05-2.00%) may be used. In some examples, a concentration of a salt of humic acid below about 0.25% may be used. In some examples, a concentration of peat of about 0.5% may be used.

[00157] Many different types of dried bacteria may be recoverable using the methods disclosed herein. Nonlimiting examples of these bacteria may include γ-proteobacteria, β- proteobacteria, a-proteobacteria, ε-proteobacteria, bacteroidetes, acinobacteria, or firmicutes, In some examples, the bacteria capable of being recovered using the methods disclosed herein include Gram-negative bacteria.

[00158] Nonlimiting examples of bacteria recoverable using the methods disclosed herein may be from the genera Acetobacter, Acinetobacter, Aeromonas, Agrobacterium, Alcaligenes, Arcobacter, Bifidobacterium, Bradyrhizobium, Burkholderia, Campylobacter, Citrobacter, Cytophaga, Enterobacter , Enter ococcus, Erwinia, Escherichia, Francisella, Helicobacter, Klebsiella, Lactobacillus, Legionella, Listeria, Oenococcus, Paracoccus, Pasteurella,

[00159] Nonlimiting examples of bacteria recoverable using the methods disclosed herein may be Acetobacter aceti, Acinetobacter calcoaceticus, Aeromonas hydrophilia, Aeromonas salmonicida, Agrobacterium tumifaciens, Alcaligenes eutrophus, Arcobacter butzleri,

[00160] In some examples, the bacteria cultured using the methods disclosed herein may not be from the order Actinomycetales (e.g., microbes from this order may be excluded). In some examples, the bacteria cultured using the methods disclosed herein may not be from the phyla Acidobacteria and Verrucomicrobia (e.g., microbes from one or both of these phyla may be excluded). In some examples, the excluded Acidobacteria may belong to subdivision 1 only. In some examples, the excluded Verrucomicrobia may belong to subdivision 4 only.

[00161] In some examples, the dried bacteria that have been recovered are enumerated. The enumerated bacteria may be counted directly. The enumerated bacteria may be calculated. In some examples of calculating unculturable bacteria, total bacteria may be enumerated (total = culturable + previously unculturable) and the number of culturable bacteria may be subtracted from the total. In some examples, a sample or dilution thereof may be cultured on agar- containing nutrient media. One of the media may contain humic acid, a salt thereof, an analog thereof, or peat. The other of the media may not contain humic acid or related substances. In the case where unculturable bacteria have been recovered on the medium containing humic acid, the bacteria enumerated on that medium are generally greater than the number of bacteria enumerated on the same medium not containing humic acid. Subtraction of the latter from the former (number on humic acid medium minus number on medium not containing humic acid) yields an estimate of the number of unculturable bacteria in the sample.

[00162] In some examples, the bacteria enumerated or counted using medium containing humic acid, salts thereof, analogs thereof, or peat, are greater than the number of bacteria enumerated using the same medium without humic acid and/or related substances. In some examples, the number of bacteria in presence of humic acid and/or related substances may be at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-,

2.7- , 2.8-, 2.9- 3.0-, 3.1-, 3.2-, 3.3-, 3.4-, 3.5-, 3.6-, 3.7-, 3.8-, 3.9-, 4.0-, 4.1-, 4.2-, 4.3-, 4.4-, 4.5- , 4.6-, 4.7-, 4.8-, 4.9-, 5.0-, 5.2-, 5.4-, 5.6-, 5.8-, 6.0-, 6.2-, 6.4-, 6.6-, 6.8-, 7.0-, 7.2-, 7.4-, 7.6-,

7.8- , 8.0-, 8.5-, 9.0-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000-fold greater than the number of bacteria obtained using medium without humic acid. Recovery of dried bacteria using the methods disclosed here may not be complete. In other words, the number of unculturable bacteria in the dried samples that are recovered using the methods may not be all of the unculturable bacteria within a population. Perhaps a fraction of the total unculturable bacteria in a bacterial population are recovered using the disclosed methods.

Example embodiments of the invention

[00163] 1. A method, comprising, consisting essentially of, or consisting of:

[00164] recovering bacteria from a dried sample, using a medium containing humic acid, a salt thereof, an analog thereof, or peat.

[00165] 2. The method of embodiment 1, including:

[00166] enumerating the bacteria recovered from the sample.

[00167] 3. The method of embodiment 2, where the enumerating step determines a number of bacteria greater than a number determined by using a medium that does not contain humic acid, a salt thereof, an analog thereof, or peat.

[00168] 4. The method of any one of embodiments 2 or 3, where a number of bacteria enumerated using medium containing humic acid, a salt thereof, an analog thereof, or peat, is greater than a number of bacteria enumerated using the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat, by at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9- 3.0-, 3.1-, 3.2-, 3.3-, 3.4- , 3.5-, 3.6-, 3.7-, 3.8-, 3.9-, 4.0-, 4.1-, 4.2-, 4.3-, 4.4-, 4.5-, 4.6-, 4.7-, 4.8-, 4.9-, 5.0-, 5.2-, 5.4-, 5.6-. 5.8-. 6.0-. 6.2-. 6.4-. 6.6-. 6.8-. 7.0-. 7.2-. 7.4-. 7.6-. 7.8-. 8.0-. 8.5-. 9.0-. 10-. 20-. 30-. 40-. 50-, 60-, 70-, 80-, 90-, or 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000- fold.

[00169] 5. The method of any one of embodiments 1-4, where the moisture content of the dried sample is less than about 50%, 40%, 30%, 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[00170] 6. The method of any one of embodiments 1-5, where the dried sample is spray dried, freeze dried, air dried, or drum dried.

[00171] 7. The method of any one of embodiments 1-6, where the dried sample is prepared as a liquid formulation prior to drying.

[00172] 8. The method of any of embodiments 1-7, where the dried sample includes a non-aqueous carrier.

[00173] 9. The method of embodiment 8, where the non-aqueous carrier includes a maltodextrin.

[00174] 10. The method of embodiment 9, where the maltodextrin has a dextrose equivalent value of about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

[00175] 11. The method of embodiment 8, where the non-aqueous carrier includes a monosaccharide, a disaccharide, sugar alcohol, or oligosaccharide.

[00176] 12. The method of any one of embodiments 1-11, where the dried sample, prior to the recovering step, is stored at a temperature of between about 20-25°C.

[00177] 13. The method of any one of embodiments 1-12, where the dried sample, prior to the recovering step, is stored at a relative humidity of between about 20-90%.

[00178] 14. The method of any one of embodiments 1-13, where the dried sample, prior to the recovering step, is stored for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 42, or 64 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 48, or 52 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, or 48 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[00179] 13. The method of any one of embodiments 1-14, where the dried sample is dissolved prior to the recovering step.

[00180] 14. The method of any one of embodiments 1-13, where the recovering step uses a CFU assay.

[00181] 15. The method of any one of embodiments 1-14, where the medium includes a gelling agent.

[00182] 16. The method of any one of embodiments 1-5, where the medium includes agar.

[00183] 17. The method of any one of embodiments 1-16, where the enumerating step includes counting colonies that form on the medium after about 6 days of incubation at about 30°C, and the medium includes agar.

[00184] 18. The method of any one of embodiments 1-17, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

[00185] 19. The method of embodiment 18, where the humic acid includes Sigma-Aldrich No. 53680 or Alfa Aesar No. 41747.

[00186] 20. The method of any one of embodiments 1-17, where a concentration of the salt of humic acid in the medium is greater than 0% and less than about 0.25% (weight/volume).

[00187] 21. The method of embodiment 20, where the salt of humic acid includes Sigma- Aldrich No. HI 6752.

[00188] 22. The method of any one of embodiments 1-21, where the bacteria recovered from the dried sample include non-spore-forming bacteria.

[00189] 23. The method of any one of embodiments 1-22, where the bacteria recovered from the dried sample include Gram-negative bacteria. [00190] 25. The method of any one of embodiments 1-23, where the bacteria recovered from the sample include Bradyrhizobia.

[00191] 26. A method for determining viable bacteria in a sample, comprising, consisting essentially of, or consisting of:

[00192] providing a spray-dried sample that contains bacteria;

[00193] plating the sample, or dilution thereof, on an agar-containing medium containing humic acid, a salt thereof, an analog thereof, or peat, such that bacterial colonies form on the medium; and

[00194] counting the bacterial colonies,

[00195] where a number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, is at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-,

1.7- , 1.8-, 1.9-, 2.0-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9- 3.0-, 3.1-, 3.2-, 3.3-, 3.4-, 3.5- , 3.6-, 3.7-, 3.8-, 3.9-, 4.0-, 4.1-, 4.2-, 4.3-, 4.4-, 4.5-, 4.6-, 4.7-, 4.8-, 4.9-, 5.0-, 5.2-, 5.4-, 5.6-,

5.8- , 6.0-, 6.2-, 6.4-, 6.6-, 6.8-, 7.0-, 7.2-, 7.4-, 7.6-, 7.8-, 8.0-, 8.5-, 9.0-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000-fold greater than a number of bacterial colonies formed when the sample or dilution is plated on the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat.

[00196] 27. The method of embodiment 26, where the spray-dried sample is stored for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 42, or 64 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 48, or 52 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, or 48 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years prior to the plating step.

[00197] 28. The method of any one of embodiments 26 or 27, where the spray-dried sample includes at least one agrochemical.

[00198] 29. The method of any one of embodiments 26-28, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume). [00199] 30. The method of embodiment 29, where the humic acid includes Sigma-Aldrich No. 53680 or Alfa Aesar No. 41747.

[00200] 31. The method of any one of embodiments 26-30, where at least some of the bacterial colonies are formed by Gram-negative bacteria.

[00201] 32. The method of any one of embodiments 26-31, where at least some of the bacterial colonies are formed by Bradyrhizobium .

[00202] 33. A method for determining viable bacteria, comprising, consisting essentially of, or consisting of:

[00203] providing a spray-dried sample that contains bacteria;

[00204] solubilizing the spray-dried sample;

[00205] plating the solubilized sample, bacteria therefrom, or a dilution thereof, on an agar-containing medium containing humic acid, a salt thereof, an analog thereof, or peat, such that bacterial colonies form on the medium; and

[00206] enumerating the bacterial colonies,

[00207] where a number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, is at least is at least about 1.1-, 1.2-, 1.3-, 1.4-, 1.5- , 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.1-, 2.2-, 2.3-, 2.4-, 2.5-, 2.6-, 2.7-, 2.8-, 2.9- 3.0-, 3.1-, 3.2-, 3.3-, 3.4-, 3.5-, 3.6-, 3.7-, 3.8-, 3.9-, 4.0-, 4.1-, 4.2-, 4.3-, 4.4-, 4.5-, 4.6-, 4.7-, 4.8-, 4.9-, 5.0-, 5.2-, 5.4-, 5.6-, 5.8-, 6.0-, 6.2-, 6.4-, 6.6-, 6.8-, 7.0-, 7.2-, 7.4-, 7.6-, 7.8-, 8.0-, 8.5-, 9.0-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, or 100-, 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-, 1000-, or 2000-fold greater than a number of bacterial colonies formed when the solubilized sample, bacteria therefrom, or dilution thereof, is plated on the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat,

[00208] where the number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, that do not form on the medium that does not contain humic acid, a salt thereof, an analog thereof, or peat, represent unculturable bacteria. [00209] 34. The method of embodiment 33, where the spray-dried sample includes at least one agrochemical.

[00210] 35. The method of any one of embodiments 33 or 34, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

[00211] 36. The method of embodiment 35, where the humic acid includes Sigma-Aldrich No. 53680 or Alfa Aesar No. 41747.

[00212] 37. The method of any one of embodiments 33-36, where at least some of the bacteria forming colonies on the medium containing humic acid, a salt thereof, an analog thereof, or peat, include Gram-negative bacteria.

[00213] 38. The method of embodiment 37, where at least some of the Gram-negative bacteria include Bradyrhizobium .

Examples

[00214] The following examples are for illustrating various embodiments and are not to be construed as limitations.

Example 1. Humic acid enhances recovery of spray-dried Bradyrhizobium japonicum

[00215] In this study, Bradyrhizobium bacteria were formulated, as described below, and then spray dried. Spray drying used a triple nozzle, where the primary formulation, containing the bacteria, was fed through an inner nozzle feed to create the particle core, and a secondary formulation was fed through an outer nozzle, to coat the bacterial core particle.

[00216] The bacteria used as the starting material was Bradyrhizobium japonicum strain 273 that had been propagated in liquid medium and then had been stored in a liquid formulation in a polypropylene bladder. A volume of material from the bladder was centrifuged and the bacterial pellet was re-suspended in one-tenth of the original volume to obtain a ten-fold concentrated culture. Various formulations containing the concentrated culture were then made with a 70:30 ratio of Maltodextrin Ml 50 to a simple sugar, where the simple sugar for each primary formulation is described in Table 1 below. Some of the formulations contained other additives, as indicated in Table 1. [00217] The concentrated culture was added to each formulation at a rate of 69% concentrated culture per weight of formulation material. The final primary formulations consisted by weight of 69% concentrated culture, 21% MALTRIN® Ml 50 (maltodextrin), 9% maltose monohydrate or sorbitol or fructose or xylitol, dependent on formulation, as noted in Table 1 below, 0.08% potassium phosphate dibasic, and 0.02% potassium phosphate monobasic. The final secondary feed formulation consisted of (by weight): 85% deionized water, 0.08% potassium phosphate dibasic, 0.02% potassium phosphate monobasic, 12.75% MALTRIN® Ml 50 (maltodextrin), and 2.25%) maltose monohydrate. Samples were incubated at 25 °C for either 1 or 24 hours as indicated in Table 1 below. All samples were spray dried using a triple nozzle with a target outlet temperature of less than 50°C. The primary feed rate used was 2mL/minute and the secondary feed rate was 11% using a BUCHI Mini Spray Dryer B-290.

[00218] Spray-dried samples were stored in each of two conditions. One condition was 54% relative humidity at room temperature. The second condition was 65% relative humidity at 25 °C. Stability testing was performed after various days in storage, as noted in Table 2 below. For each time point, the spray-dried samples were re-suspended in standard phosphate buffer solution and serially diluted in 96-well plates to obtain a final dilution range of 10^"2-10^"7.

[00219] Dilutions were plated onto both YEM agar (where lOg/L mannitol, 0.5g/L yeast extract, O. lg/L sodium chloride, 0.5g/L potassium phosphate dibasic anhydrous, 0.2g/L magnesium sulfate heptahydrate, 12g/L granulated agar were autoclaved and lOOmg/L polymixin B sulfate was added post autoclaving prior to pouring plates) and YEMHA agar (made the same as YEM agar but, prior to autoclaving, included an addition of 2.5g/L humic acid No. 53680; Sigma-Aldrich, St. Louis, Missouri, USA). Plates were placed in sealed plastic bags and incubated at 30°C for six days or until colonies were visible under a microscope.

Colonies were manually counted and colony forming units (CFU)/mL were calculated. Results for each stability test are found in Tables 2 and 3 below. The recovery results for each formulation stored at 54% relative humidity and room temperature are found in Table 2 below while the results for formulations stored at 65% relative humidity and 25 °C are found in Table 3 below.

Table 2: Comparison of Bradyrhizobium japonicum recovery under 54% relative humidity and room temperature conditions in various formulations using YEM and YEMHA agar

YEM YEMHA

Formulation 0 da ys 13 days 28 days 64 days 0 days 1 3 days 28 days 64 days

4.63E+10 9. 50E+09 1.99E+09 1.00E+04

D18.1 4.63E +10 5.13E+0 3 1.01E+09 l.OOE+0 I

(1.0) (1.9) (2.0) (1.0)

3.40E+10 8. 30E+09 7.85E+08 1.00E+04

D18.2 3.40E +10 4.73E+0 3 3.15E+08 l.OOE+0 I

(1.0) (1.9) (2.5) (1.0)

4.05E+10 7. 30E+09 4.90E+08 1.00E+04

D18.3 4.05E +10 4.48E+0 3 1.35E+08 l.OOE+0 I

(1.0) (1.6) (3.6) (1.0)

3.38E+10 9. 35E+09 1.14E+07 1.00E+04

D18.4 3.38E +10 4.40E+0 i 3.18E+06 l.OOE+0 I

(1.0) 20.6) (3.6) (1.0)

(1.0) (5.3) (28.8)

Values in parenthesis represent normalized data where each CFU/mL count on YEMHA agar was compared to the corresponding CFU/mL count on YEM agar.

[00220] In general, the data showed that recovery of Bradyrhizobium japonicum in spray dried formulations was higher when grown on agar media with humic acid, YEMHA, than when grown on media without humic acid, YEM. This effect was noticed across various formulations over varying amounts of time post spray dry.

[00221] While example compositions, methods, and so on have been illustrated by description, and while the descriptions are in considerable detail, it is not the intention of the applicant to restrict or in any way limit the scope of the application. It is, of course, not possible to describe every conceivable combination of components or methodologies for purposes of describing the compositions, methods, and so on described herein. Additional advantages and modifications will readily appear to those skilled in the art. Therefore, the invention is not limited to the specific details and illustrative examples shown and described. Thus, this application is intended to embrace alterations, modifications, and variations that fall within the scope of the application. Furthermore, the preceding description is not meant to limit the scope of the invention.

[00222] To the extent that the term "includes" or "including" is employed in the detailed description or the claims, it is intended to be inclusive in a manner similar to the term

"comprising" as that term is interpreted when employed as a transitional word in a claim.

Furthermore, to the extent that the term "or" is employed in the detailed description or claims (e.g., A or B) it is intended to mean "A or B or both". When the applicant intends to indicate "only A or B but not both" then the term "only A or B but not both" will be employed. Thus, use of the term "or" herein is the inclusive, and not the exclusive use. See, Bryan A. Garner, A Dictionary of Modern Legal Usage 624 (2d. Ed. 1995). Also, to the extent that the terms "in" or "into" are used in the specification or the claims, it is intended to additionally mean "on" or "onto." Furthermore, to the extent the term "connect" is used in the specification or claims, it is intended to mean not only "directly connected to," but also "indirectly connected to" such as connected through another component or components.

Claims

CLAIMS We claim:

1. A method for determining viable bacteria in a sample, comprising:

providing a dried sample that contains bacteria;

plating the sample, or a dilution thereof, on an agar-containing medium containing humic acid, a salt thereof, an analog thereof, or peat, such that bacterial colonies form on the medium; and

counting the bacterial colonies,

where a number of bacterial colonies formed on the medium containing humic acid, a salt thereof, an analog thereof, or peat, is at least about 1.2-fold greater than a number of bacterial colonies formed when the sample or dilution is plated on the same medium that does not contain humic acid, a salt thereof, an analog thereof, or peat.

2. The method of claim 1, where a moisture content of the dried sample is less than about 50%, 40%, 30%, 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

3. The method of any one of claims 1-2, where the dried sample has been spray dried, freeze dried, air dried, or drum dried.

4. The method of any one of claims 1-3, where the dried sample includes at least one agrochemical.

5. The method of any one of claims 1-4, where the dried sample has been stored for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28, 30, 42, or 64 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20, 24, 48, or 52 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, or 48 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years prior to the plating step.

6. The method of any one of claims 1-5, where a concentration of the humic acid in the medium is greater than 0% and less than about 5% (weight/volume).

7. The method of any one of claims 1-5, where a concentration of the salt of humic acid in the medium is greater than 0% and less than about 0.25% (weight/volume).

8. The method of any one of claims 1-7, where at least some of the bacterial colonies are formed by non-spore-forming bacteria.

9. The method of any one of claims 1-8, where at least some of the bacterial colonies are formed by Gram-negative bacteria.

10. The method of any one of claims 1-9, where at least some of the bacterial colonies are formed by Bradyrhizobium .

11. The method of any one of claims 1-10, where the dried sample is prepared as a liquid formulation prior to drying.